Capturing Cellular Dynamics with Millisecond Temporal Resolution

Coupling Light Stimulation and High Pressure Freezing

May 12, 2014

The combination of two powerful techniques: optogenetics and high-pressure freezing now makes it possible to visualize a dynamic cellular activity with temporal resolution of 5 milliseconds. By coupling a flash of light with high-pressure freezing, the process of vesicle recycling at the synapses can now be imaged by electron microscopy.

Optogenetics combined with high pressure freezing

Traditionally electron microscopy only captures a "snap-shot" of a cell. But a snap-shot can be deceiving. Consider for a moment this photo. Is he falling?  Floating? Or flying?

To understand dynamics of any process, one must know what came before and what came after. Looking at the micrograph below one might argue what is this particular cell doing.

Fig. 1: Micrograph showing fusing vesicles in the active zone. Arrow indicated fusing vesicle at a specific time after light stimulation –15 ms. PSD, postsynaptic density.

Is a vesicle fusing?  Or is the cell removing membrane from the surface?  What is the sequence of events that takes place in the particular cell? Such information cannot be extracted from random static images. However, by collecting images at a later time point (at 30 ms), we can demonstrate that the cell is in fact expelling the contents of vesicles.

Figure 2: Micrograph showing fusing vesicles in the active zone. Arrow indicated fusing vesicle at a specific time after light stimulation –30 ms.

Employing optogenetic tools a flash of light can trigger a dynamic cellular event. For example, by using the light activated ion channel (channelrhodopsin) a synaptic transmission can be stimulated in a neuron. By alignment of the triggered light stimulation with high-pressure freezing, regulated vesicle fusion and the subsequent vesicle recycling at synapses can be subsequently visualized in electron micrographs. By analyzing a sequence of images at specific time points, the progression of events can be assembled.

Flash-and-freeze approach can be expanded to other cellular events. A variety of light-sensitive tools are becoming available to stimulate particular cellular activities.

Optogenetics in combination with high pressure freezing adds another dimension to electron microscopy – time.

…. And if you are still curious, the ski acrobat was in fact flying.

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